3.3. Expatiated Pathophysiological Heterogeneity in ΔKPQ
3.3.2. Flecainide Exacerbates Intrinsic Heterogeneity in ΔKPQ
One of the arrhythmogenic mechanisms proposed for the LQTS substrate is amplification of the SDR, which when present in either early or late phases of the AP can lead to the development of a variety of arrhythmias403. Amplification of intrinsic heterogeneity together with premature impulses could principally underlie the mechanistic trigger for ventricular arrhythmias in cardiac pathologies such as heart failure271 and “torsade de pointes” in LQTS3406. Gene-‐specific therapy includes a 2-‐hit approach: The first is linking the genetic mutation to the ionic channel dysfunction, whereas the second is identifying the pharmacological agents that can modulate the mutated channels in a differential manner281.
The response of arrhythmogenic LQTS3 substrate to AADs remains the subject of strenuous debate.
Schwartz et al. and others reported that NaV1.5 blocker Mexiletine (a Class IB AAD) is more effective in shortening the QT-‐interval in LQTS3 patients than other LQTS due to K+-‐channel mutations282, 574. Shimizu et al. showed that a K+ channel opening agent (Nicorandil) abbreviated the QT-‐interval and the monophasic AP duration in LQTS1 patients281. Shortening of QT interval isn’t necessarily harmonious with decreasing or preventing arrhythmia risk in these patients281, since a direct validation of the QT interval as a measure of APD spatial dispersion is still lacking575. On the other hand, NaV1.5 blocking agents haven’t been reported to be ineffective in treating LQTS3 only576, but also dangerously proarrhythmic570, sometimes despite a normalized QT-‐interval on ECG577. Hence normalizing the QT-‐interval is not necessarily congruent with decreased SCD risk.
We show in the following results that exaggerated SDR is predominantly observed in the ΔKPQ mouse upon exposure to Flecainide at clinically valid concentrations, which could make it a plausible mechanism in increasing arrhythmia susceptibility in LQTS3 patients managed with NaV1.5 blockers.
Expatiated Pathophysiological Heterogeneity in ΔKPQ 109
In Figure 39, APs and their corresponding APD dispersion maps (APD25 (a), APD50 (b)) for WT and ΔKPQ at t0 and t5 of Flecainide [1μM] are shown. As previously described, the same mask of the LV free wall was repeatedly used for the same prep to reproduce the maps at different time points of exposure to the drug. The APs on the left are chosen from the located points in the maps where white dots reside along the longitudinal direction with corresponding blue colored APs, and red dots along the transversal direction with corresponding red colored APs. Measurements were conducted at t0, t1, t3 and t5, but only t0 and t5 are shown in the figure. The morphological changes in OAPs are tracked by superimposing the APs at t5 over their control AP (in dotted line). APWT at t0 perfectly overlap, and their corresponding APD25 map further confirms the homogeneity of the cardiac tissue under these conditions. The map’s associated histograms are shown in Figure 40a (leftmost panel). The unimodal distribution of APD25,WT values are centered around 7.2ms with a dispersion of ΔAPD25,WT = 7.9ms. The average dispersion in the WT group (n=4, Figure 41) under control conditions is 8.7±1.7ms. In contrast, the APD25,ΔKPQ map (Figure 39a, below left) shows patchy regions of more prolonged APs across the LV free wall (lighter blue), with reduced APs found mainly at the basal side. The distribution of the APD25,ΔKPQ values is centered around 13.5ms, has a wider base than its WT counterpart with ΔAPD25,ΔKPQ = 16.2ms (Figure 40a, right). In conclusion, at t0 when Flecainide is not yet in perfusion, not only APD25,ΔKPQ in terms of values is significantly larger (Figure 42a, right and left panels), but also ΔAPD25,ΔKPQ (n=4) is significantly wider than its WT counterpart (ΔAPD25,WT = 8.7±1.7ms; ΔAPD25,ΔKPQ = 14.9±4.1ms, p-‐value<0.05, Figure 41), corresponding to the appearance of sketchy clusters of longer APDs in the mutated substrate.
At t5, the APWT have shallower upstrokes with a decreased notch, followed by an increase in the APD mainly at later repolarization phases. While the map shows a homogenous prolongation in APD25,WT values, the histograms at t5 (Figure 40a, left) conserve a high peak centered at 10.2ms with a longer tail, causing an increase in base width (ΔAPD25,WT = 17.4ms in this example). On average, ΔAPD25,WT increased in total of ~120% to reach a value of 19.7±5.1ms by t5 (Figure 41). The response of the ΔKPQ tissue to Flecainide at t5 however appears more dramatic, with multiple adjacent regions of shorter, intermediate, and prolonged APD25 allocated between the base and apex of the LV free wall (Figure 39a, bottom, right). On the left side of the map, the APs selected from 4 different locations within the ventricle display gradual loss of their notch, followed by a proportional increase in the APD. By looking at the position of each of the APs, it becomes obvious that epicardial cells that once displayed almost superimposable APs at t0 (for instance locations 1 and 3, along the longitudinal direction) are separated by an APD25 gradient exceeding 30ms after 5min exposure to Flecainide. A direct comparison of the APD25,ΔKPQ maps at t0 and t5 reveals the possibility that the patterns formed at t5 may be the result of amplification of intrinsic heterogeneity found within the mapped epicardial layer, indicated by the patchy arrangement of APD25 initially found at t0. Nevertheless, it’s not yet clear whether the patterns that appeared with Flecainide can potentially be predicted from the initial conditions at t0. The histograms of the corresponding map no longer display any particular peak and nicely span a whole range of values with a dispersion ΔAPD25,ΔKPQ = 35.7ms (Figure 40a, right). From the bar graphs in Figure 41, it becomes evident that APD25 dispersion is significantly larger in the ΔKPQ substrate at either t0 or t5, with an increase of ~120% and ~135% in ΔAPD25,WT and ΔAPD25,ΔKPQ respectively between t0 and t5 (Figure 43).
The effects observed in APD25 are translated to later phases of the AP (Figure 39b). The circular becomes statistically significant (Figure 41). Counterintuitively, the dispersion in APD50 values are considerably larger in the WT group, despite the presence of symmetry breaking exclusively in the the substantial widening of the distribution base), where ΔAPD25,WT =17.4ms. The loss indicating a considerable loss of previously existing higher (dF/dt)max at t0.
Expatiated Pathophysiological Heterogeneity in ΔKPQ 111
While some heterogeneity is still observed at repolarization levels beyond 50%, it’s less prominent than the one detected at 25% and 50% (maps not shown here). The dispersion of APD75 values (Figure 41) is not significantly different between the two groups at t0 (ΔAPD75,WT =24.6±4.3ms;
ΔAPD75,ΔKPQ = 28.4±8.3ms; p-‐value=0.44), nor at t5 (ΔAPD75,WT = 33.9±7.2ms; ΔAPD75,ΔKPQ = 30.2±3.3ms; p-‐value=0.39).
In addition to dispersion analysis, APDs at different levels of repolarization were analyzed and compared between WT and ΔKPQ at different time points of Flecainide exposure (Figure 42). While APD25, APD50 and APD75 show statistically significant differences at control conditions (t0) between the experimental groups, exposure to Flecainide caused a consistent prolongation of all APDs abolishing at almost all time points the existing differences between them.
The effect AP abbreviation or prolongation has on spatial dispersion has not been quantitatively well determined. It’s strongly believed however that asymmetrical AP prolongation in the heart leads to increased spatial dispersion, enhancing medium susceptibility for arrhythmic activity. From my current data, the correlation between APDxx and ΔAPDxx (xx standing for any percentage repolarization) could by itself provide a quantitative indicator for the possible occurrence of symmetry breaking in the ventricle. Following the previous analysis of ΔAPD (Figure 41), APD values were compared between WT (N=8) and ΔKPQ (N=8). APD25 (along longitudinal and transversal directions, Figure 42a), APD50 (longitudinal only, Figure 42b), APD75 (longitudinal only, Figure 42c) bar graphs are displayed. The ΔKPQ heart for all APDs at t0 shows significantly larger values than its WT counterpart. For instance, APD25,WT (Long.) = 6.0±1.0ms, APD25,ΔKPQ (Long.) = 10.0±4.1ms (p-‐value <0.05). The transversal APD25 are particularly shown here because of their distinct changes with Flecainide (when comparing the groups) relatively to the longitudinal values, which is not the case for the APD50 or APD75 therefore only longitudinal values are displayed in the figure. Similarly, APD50,WT = 27.7±5.6ms, APD50,ΔKPQ = 36.1±6.1ms (p-‐value<0.05), whereas APD75,WT = 46.7±6.9ms, APD75,ΔKPQ = 54.2±5.7ms (p-‐value<0.05).
Figure 41. Effects of Flecainide on APD dispersion for WT and ΔKPQ at 25%, 50% and 75%
AP repolarization. APDxx is calculated for each prep at t0 and t5 as the difference between the 95th and 5th percentile of APDxx distribution. The difference in ΔAPD25 at t0 is further amplified with Flecainide, by an increase of 120% and 135% for WT and ΔKPQ respectively. Despite this latter comparable increase, symmety breaking occurs only in the mutated heart at this experimental time point. The increase of ~45% in ΔAPD50,WT vs. ~5% in ΔAPD50,ΔKPQ changed the statistics at t5. The changes in ΔAPD75 remain statistically insignificant between the 2 groups. Annotations in figure:
* (p-‐value<0.05); ** (p-‐
value<0.01); n.s. not significant.
As all APDs prolong with Flecainide in both groups (WT and ΔKPQ), the maximal increase is naturally observed in APD25, being the initially smallest entity measured. The total increase in APD25 along both directions is not only compared among the groups, but also contrasted to the increase in ΔAPD25 between t0 and t5 (Figure 43). To recall from previous experiments done on the mdx heart with its WT control, symmetry breaking occurs only in the WT heart at t10 but didn’t take place in the mdx heart. The total increase (percentage wise) in APD25 was significantly larger in WT (summarized in Figure 43) in either direction compared to the changes detected in the mdx.
Contrastingly, comparing the percentage prolongation in APD25 at t5 between ΔKPQ and WT, the latter has shown a considerably larger increase along the longitudinal direction (the transversal APD25 increase is 100% for either group), yet symmetry breaking took place in the ΔKPQ ventricle and not the WT. Therefore, it seems that the extent of APD prolongation between control and end of treatment conditions alone cannot predict loss of symmetry in APD patterns.
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Figure 42. Bar graphs showing the progression of APD25 (a), APD50 (b) and APD75 (c) with Flecainide in both WT (N=8) and ΔKPQ (N=8). Only for the APD25, both longitudinal (left) and transversal (right) are shown. For the remaining APD50 and APD75, bar graphs contain values picked along the longitudinal direction only. At initial conditions, the ΔKPQ substrate shows a consistent prolongation of its APDs compared to its WT counterpart. As Flecainide proceeds to 5min,, the significant difference between the groups is abolished except in APD25 (transversally), where the APDs remain significantly higher in the mutated at all experimental time points. The overall increase in APD values between t0 and t5 can be summarized as such: (Long.) 150% in APD25,WT vs. 105% in APD25,ΔKPQ. (Trans.) 100% in both APD25,WT and APD25,ΔKPQ . 55% in APD50,WT vs. 43% in APD50,ΔKPQ. 33% in APD75,WT vs. 30% in APD75,ΔKPQ.
Expatiated Pathophysiological Heterogeneity in ΔKPQ 113 could bring forward particular requirements that might expedite its appearance.
dispersion of repolarization in small hearts, where electrotonic conduction is normally sufficiently high to smooth out functional heterogeneities. Such patterns formation in larger hearts, where more structural heterogeneity prevails, could be several orders of magnitude more dangerous and lethal.
In cardiac tissues, where electrical excitation and conduction across coupled cells is maintained by Na+, pharmacological interventions through NaV1.5 blocking have the potential to amplify cardiac instability by decoupling universal parameters of excitability and conductivity, including weakening electrical depolarization, slowing conduction, increasing repolarization time and finally splitting the intact tissue into adjacent zones of steep repolarization gradients.
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Patchy distribution of prolonged APDs prevents a congruent repolarization and increases spatial-‐functional heterogeneity. Scale bar = 1mm.
Expatiated Pathophysiological Heterogeneity in ΔKPQ 115
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From a point stimulation (pacing electrode remaining at exact same position as in Figure 44), activation is slower and asymmetrical on the apico-‐
basal axis. Multiple adjacent zones of inconsistent repolarization properties amplify spatial-‐temporal heterogeneity and SDR, rendering the tissue a highly susceptible substrate for arrhythmia. Scale bar = 1mm.
x-axis y-axis Slope ± error Correlation
coef. (R!)
LSEF PF 0.55±0.17 0.41
LESF PF 0.59±0.20 0.41
PF AF 0.97±0.14 0.75
LSEF PF 0.67±0.15 0.58
LESF PF 0.75±0.14 0.64
PF AF 0.97±0.12 0.82
LSEF PF 0.87±0.05 0.90
LESF PF 0.89±0.05 0.92
PF AF 0.99±0.03 0.97
LSEF PF 0.66±0.07 0.86
LESF PF 0.97±0.11 0.84
PF AF 1.33±0.17 0.82
LSEF PF 0.72±0.13 0.67
LESF PF 0.97±0.20 0.62
PF AF 1.20±0.20 0.75
LSEF PF 0.57±0.08 0.59
LESF PF 0.91±0.06 0.87
PF AF 0.99±0.15 0.65
Longitudinal CVTransversal CV WTmdx MergedWTmdx Merged
Figure 46. Table summarizing the slopes and determination coefficients (R2) extracted from the linear regressions of the velocities (analysis done in Figure 15). The methods’ outcomes being Vmax (Longitudinal CV) and Vmin (Transversal CV) are initially separated into two categories. The methods are plotted against each other for a direct comparison. The substrates (WT and mdx) being two different entities were treated each aside first, then merged. R2 consistently scored higher values in merged sets (linear regressions in Figure 15 contain the merged data points) than in each set aside particularly for Vmax values. The slopes of merged sets are closer to unity (in blue) except for the value in pink, where the slope is not close to one, but associated with a low R2 value as well.
Values in red show sporadic unity slopes and in green slopes higher than one occurring when each of the WT and mdx sets are treated separately.
Expatiated Pathophysiological Heterogeneity in ΔKPQ 117
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Chapter 4
Discussion
Microscopic components derived from active ionic and passive membrane properties, and macroscopic discontinuities reflecting branching anatomical features or other structural heterogeneities, can produce changes in the shape of the AP and CV, characteristics of cardiac conduction that cannot be predicted by continuous propagation models256. Discontinuous propagation implies that a delicate local source-‐sink balance governs successful conduction across the tissue, where the amount of charges supplied by the source proximally must at least be equal to the charges required to excite the cardiac membrane at the sink distally257. At the cellular level, this is controlled by membrane excitability followed by the state of electrical coupling between cells. In the ventricular myocardium, the principal active determinant of excitability is probably the cardiac NaV1.5lxviii, where the magnitude of INa,f plays a decisive role in the subsequent propagation of the electrical wave from the source location further down the multicellular network. Meticulous opening and closing of the cardiac ion channels results in the generation of the AP, ensures its successful propagation, maintains the intricate coupling of electrical and mechanical activities and orchestrates the sequence of ionic channels to bring about the lucrative termination of the AP260. Henceforth, abnormalities of NaV1.5 expression, regulation or kinetics will translate into cardiac instabilities that induce electrical vulnerability and precipitate rhythm disturbances. How could NaV1.5 perturbations influence the stability of the entire cardiac tissue?
In this thesis work, two models of NaV1.5 abnormalities were investigated, and further modulation of NaV1.5 was resumed through pharmacological interventions. The results of this thesis have tried to answer the questions proposed in the last section of the Introductionlxix by:
1. Characterizing electrical instabilities in conduction in a model where NaV1.5 is lost exclusively from the LM of the cardiomyocyte.
2. Implementing and validating different analytical strategies to evaluate conduction velocity in a medium with anisotropic and atypical spatial-‐temporal patterns of activation.
3. Investigating a circumstantiated proarrhythmic mechanism of Flecainide (NaV1.5 blocker) in normal heart tissues using clinically valid concentrations.
4. Providing adminicular evidence that a model harboring a LQTS3 mutation is exceedingly destabilized in the presence of Flecainide.
lxviii Detailed description of the two models of propagation is found in the Introduction section 1.3.1.
lxix Refer to Introduction section 1.6